Process for the production of 7-ADCA via expandase activity on penicillin G

ABSTRACT

A process is taught for the preparation and recovery of 7-aminodesacetoxycephalosporanic acid (7-ADCA) via enzymatic ring expansion activity on penicillin G, using a Penicillium chrysogenum transformant strain expressing expandase.

FIELD OF THE INVENTION AND BRIEF DESCRIPTION OF THE PRIOR ART

The present invention concerns a biosynthetic process for preparationand recovery of 7-aminodesacetoxycephalosporanic acid (7-ADCA).

β-Lactam antibiotics constitute the most important group of antibioticcompounds, with a long history of clinical use. Among this group, theprominent ones are the penicillins and cephalosporins. These compoundsare naturally produced by the filamentous fungi Penicillium chrysogenumand Acremonium chrysogenum, respectively.

As a result of classical strain improvement techniques, the productionlevels of the antibiotics in Penicillium chrysogenum and Acremoniumchrysogenum have increased dramatically over the past decades. With theincreasing knowledge of the biosynthetic pathways leading to penicillinsand cephalosporins, and the advent of recombinant DNA technology, newtools for the improvement of production strains and for the in vivoderivatization of the compounds have become available.

Most enzymes involved in β-lactam biosynthesis have been identified andtheir corresponding genes been cloned, as can be found in Ingolia andQueener, Med. Res. Rev. 9 (1989), 245-264 (biosynthesis route andenzymes), and Aharonowitz, Cohen, and Martin, Ann. Rev. Microbiol. 46(1992), 461-495 (gene cloning).

The first two steps in the biosynthesis of penicillin in P. chrysogenumare the condensation of the three amino acidsL-5-amino-5-carboxypentanoic acid (L-α-aminoadipic acid) (A), L-cysteine(C) and L-valine (V) into the tripeptide LLD-ACV, followed bycyclization of this tripeptide to form isopenicillin N. This compoundcontains the typical β-lactam structure.

The third step involves the exchange of the hydrophillic side chain ofL-5-amino-5-carboxypentanoic acid by a hydrophobic side chain by theaction of the enzyme acyltransferase (AT). The enzymatic exchangereaction mediated by AT takes place inside a cellular organelle, themicrobody, as has been described in EP-A-0448180.

Cephalosporins are much more expensive than penicillins. One reason isthat some cephalosporins (e.g. cephalexin) are made from penicillins bya number of chemical conversions. Another reason is that, so far, onlycephalosporins with a D-5-amino-5-carboxypentanoyl side chain could befermented. Cephalosporin C, by far the most important starting materialin this respect, is very soluble in water at any pH, thus implyinglengthy and costly isolation processes using cumbersome and expensivecolumn technology. Cephalosporin C obtained in this way has to beconverted into therapeutically used cephalosporins by a number ofchemical and enzymatic conversions.

The methods currently favoured in industry to prepare the intermediate7-ADCA involve complex chemical steps leading to the expansion andderivatization of penicillin G. One of the necessary chemical steps toproduce 7-ADCA involves the expansion of the 5-membered penicillin ringstructure to a 6-membered cephalosporin ring structure (see for instanceU.S. Pat. No. 4,003,894). This complex chemical processing is bothexpensive and noxious to the environment.

Consequently, there is a great desire to replace such chemical processeswith enzymatical reactions such as enzymatic catalysis, preferablyduring fermentation. A key to the replacement of the chemical expansionprocess by a biological process is the central enzyme in thecephalosporin biosynthetic pathway, deacetoxycephalosporin C synthetase,or expandase.

The expandase enzyme from the bacterium Streptomyces clavuligerus wasfound to carry out in vitro, in some cases, penicillin ring expansions(Baldwin et al., Tetrahedron 43(13), 3009 (1987)). In Cantwell et al.(Current Genetics, 17, 213-221 (1990)), expression of S. clavuligerusexpandase in P. chrysogenum is described. Espression of the expandasedid not result in formation of cephalosporins in a fermentation assuggested in the publications. Only when introduced into P. chrysogenumtogether with the isopenicillin N epimerase gene of S. clavuligerus,conversion of the penicillin ring structure of penicillin N (its naturalsubstrate) into the cephalosporin ring structure ofdesacetoxycephalosporin C (its natural product) was observed, asdescribed in Cantwell et al., Proc. R. Soc. Lond. B. 248 (1992),283-289. The expandase enzyme has been well characterized (EP-A-0366354)both biochemically and functionally, as has its corresponding gene. Bothphysical maps of the cefE gene (EP-A-0341892), DNA sequence andtransformation studies in P. chrysogenum with cefE have been described.

Another source for a ring expansion enzyme is the bacterium Nocardialactamdurans (formerly Streptomyces lactamdurans). Both the biochemicalproperties of the enzyme and the DNA sequence of the gene have beendescribed (Cortes et al., J. Gen. Microbiol. 133 (1987), 3165-3174; andCoque et al., Mol. Gen. Genet. 236 (1993), 453-458, respectively).

Since the expandase catalyses the expansion of the 5-memberedthiazolidine ring of penicillin N to the 6-membered dihydrothiazine ringof deacetoxycephalosporin C this enzyme would be of course a logicalcandidate to replace the ring expansion steps of the chemical process.Unfortunately, the enzyme works on the penicillin N intermediate of thecephalosporin biosynthetic pathway, but not on the readily availableinexpensive penicillins as produced by P. chrysogenum, includingpenicillin G. Penicillin N is commercially not available and even whenexpanded, its D-aminoadipyl side chain cannot be removed easily bypenicillin acylases.

It has recently been found that the expandase enzyme is capable ofexpanding penicillins with particular side chains to the corresponding7-ADCA derivative. In EP-A-268343 an in vitro process of the expansionof a penicillin with a 3-carboxyphenylacetyl or adipoyl side chain byapplying deacetoyxycephalosporin C synthetase has been described.Furthermore, this feature of the expandase has been exploited in thetechnology as disclosed in EP-A-0532341, EP-A-0540210, WO95/04148 andWO95/04149. In these disclosures the conventional chemical conversion ofpenicillin G to 7-ADCA has been replaced by the in vivo conversion ofcertain 6-aminopenicillanic acid (6-APA) derivatives in recombinantPenicillium chrysogenum strains containing an expandase gene.

More particularly, EP-A-0532341 teaches the in vivo use of the expandaseenzyme in P. chrysogenum, in combination with a 5-carboxypentanoyl sidechain as a feedstock, which is a substrate for the acyltransferaseenzyme in P. chrysogenum. This leads to the formation of5-carboxypentanoyl-6-APA, which is converted by an expandase enzymeintroduced into the P. chrysogenum strain to yield5-carboxypentanoyl-7-ADCA. Finally, the removal of the5-carboxypentanoyl side chain is suggested, yielding 7-ADCA as a finalproduct.

In WO95/04148 and WO95/04149 it has been disclosed that3'-carboxymethylthiopropionic acid and 3,3'-thiodipropionic acid,respectively were found to be substrates for the expandase, yielding2-(carboxyethylthio)acetyl- and 3-(carboxymethylthio)propionyl-7-ADCA.

However, the process of the present invention provides more advantages,because of the high pen G synthese capacity of penicillin producingstrains and the more favorable process of extraction ofphenylacetyl-7-ADCA acid. Furthermore the phenylacetyl side chain ofpenicillin G is very amenable to enzymatic cleavage, by penicillin Gamidases produced by several types of microorganisms yielding 6-APA, forinstance separase G as disclosed in EP-A-0453047.

Various publications have reported the expandase not to acceptpenicillin G as a substrate for expansion (Baldwin & Abraham (1988),Natural Product Reports, 5(2), p.129-145; Maeda et al. (1995), Enzymeand Microbial Technology, 17, 231-234; Crawford et al. (1995),Bio/technology, 13, p.58-61; Wu-Kuang Yeh et al., in 50 years PenicillinApplication (editors Kleinkauf and Von Dohren), 209 (1991), seeespecially table 3A).

Surprisingly, however, it has now been found that penicillin G producingP. chrysogenum transformed with an expandase encoding gene is capable ofproducing phenylacetyl-desacetoxycephalosporanic acid.

SUMMARY OF THE INVENTION

The present invention provides a process for the preparation andrecovery of 7-aminodesacetoxycephalosporanic acid (7-ADCA) by:

a) transforming a Penicillium chrysogenum strain with an expandase gene,under the transcriptional and translational regulation of fungalexpression signals;

b) fermenting said strain in a culture medium and adding to said culturemedium phenylacetic acid or a salt or ester thereof suitable to yieldpenicillin G, which is expanded to form phenylacetyl-7-ADCA;

c) recovering the phenylacetyl-7-ADCA from the fermentation broth;

d) deacylating phenylacetyl-7-ADCA; and

e) recovering the crystalline 7-ADCA.

Preferably, step (e) is a filtration step.

Preferably, phenylacetyl-7-ADCA is recovered from the fermentation brothby extracting the broth filtrate with an organic solvent immiscible withwater at a pH of lower than about 4.5 and back-extracting the same withwater at a pH between 4 and 10.

Moreover, a recombinant DNA vector comprising the DNA encodingexpandase, functionally linked to the transcriptional and translationalcontrol elements of a fungal gene, for instance Aspergillus nidulansgpdA gene, and the Aspergillus niger glcA gene and host cellstransformed with the same, are provided.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns the use of functional gene constructs inP. chrysogenum for the in vivo expansion of the penicillin G ringstructure to form a derivative of a key intermediate in thecephalosporin biosynthesis, 7-aminodesacetoxycephalosporanic acid, or7-ADCA. This derivative has a chemical composition so as to allowefficient solvent extraction, thus providing an economically attractiverecovery process.

Transformation of P. chrysogenum can, in principle, be achieved bydifferent means of DNA delivery, like PEG-Ca mediated protoplast uptake,electroporation or particle gun techniques, and selection oftransformants. See for example Van den Hondel en Punt, Gene Transfer andVector Development for Filamentous Fungi, in: Applied Molecular Geneticsof Fungi (Peberdy, Laten, Ogden, Bennett, eds.), Cambridge UniversityPress (1991). The application of dominant and non-dominant selectionmarkers has been described (Van den Hondel, supra). Selection markers ofboth homologous (P. chrysogenum derived) and heterologous (non-P.chrysogenum derived) origin have been described (Gouka et al., J.Biotechnol. 20 (1991), 189-200).

The application of the different transformant selection markers,homologous or heterologous, in the presence or absence of vectorsequences, physically linked or not to the non-selectable DNA, in theselection of transformants are well known.

The ring-expansion reaction, mediated by the expandase enzyme isintroduced into and expressed in this way in P. chrysogenum, forinstance in strain Panlabs P14-B10, DS 18541 (deposited at CBS underaccession number 455.95). It will be clear that in case thering-expansion reaction is carried out in mutants thereof, the mediumconditions have to be slightly adapted to obtain an efficient growth.

Furthermore, the cefE gene is placed under the transcriptional andtranslational control of fungal (be they filamentous or not) genecontrol elements, preferably derived of the P. chrysogenum gene Y(described in EP-A-0549062), the P. chrysogenum IPNS gene, the β tubulingene, the Aspergillus nidulans gpdA gene, or the Aspergillus niger glcAgene.

In summary, the present invention teaches how the activity of anexpandase enzyme introduced into P. chrysogenum can be dedicated in vivoto the ring expansion of penicillin G.

In accordance with the present invention the β-lactam intermediatephenylacetyl-7-ADCA is produced in P. chrysogenum by adding phenylaceticacid or a salt or an ester thereof to the medium. Suitable salts are forinstance those of sodium or potassium. 7-ADCA is efficiently recoveredfrom the medium through a simple solvent extraction, for instance, asfollows:

The broth is filtered and an organic solvent immiscible with water isadded to the filtrate. The pH is adjusted in order to extract thecephalosporin from the aqueous layer. The pH range has to be lower than4.5; preferably between 4 and 1, more preferably between 2 and 1. Inthis way the cephalosporin is separated from many other impuritiespresent in the fermentation broth. Preferably a small volume of organicsolvent is used, giving a concentrated solution of the cephalosporin, soachieving reduction of the volumetric flow rates. A second possibilityis whole broth extraction at a pH of 4 or lower. Preferably the broth isextracted between 4 and 1 with an organic solvent immiscible with water.

Any solvent that does not interfere with the cephalosporin molecule canbe used. Suitable solvents are, for instance, butyl acetate, ethylacetate, methyl isobutyl ketone, alcohols like butanol etc. Preferablybutylacetate is used.

Hereafter the cephalosporin is back extracted with water at a pH between4 and 10, preferably between 6 and 9. Again the final volume is reduceddrastically. The recovery can be carried out at temperatures between 0and 50° C., and preferably at ambient temperatures.

The aqueous cephalosporin solution thus obtained is treated with asuitable enzyme in order to remove the phenylacetyl side chain andobtain the desired 7-ADCA. A suitable enzyme for the same is thepenicillin G acylase as described in EP-A-0453047, also named penicillinamidase.

Preferably, an immobilized enzyme is used, in order to be able to usethe enzyme repeatedly. The methodology for the preparation of suchparticles and the immobilization of the enzymes have been describedextensively in EP-A-0222462. The pH of the aqueous solution has a valueof, for example pH 4 to pH 9, at which the degradation reaction ofcephalosporin is minimized and the desired conversion with the enzyme isoptimized. Thus, the enzyme is added to the aqueous cephalosporinsolution while maintaining the pH at the appropriate level by, forinstance, adding an inorganic base, such as a potassium hydroxidesolution, or applying a cation exchange resin. When the reaction iscompleted the immobilized enzyme is removed by filtration. Anotherpossibility is the application of the immobilized enzyme in a fixed orfluidized bed column, or using the enzyme in solution and removing theproducts by membrane filtration. Subsequently, the reaction mixture isacidified in the presence of an organic solvent immiscible with water.

After adjusting the pH to about 0.1 to 1.5, the layers are separated andthe pH of the aqueous layer is adjusted to 2 to 5. The crystalline7-ADCA is then filtered off.

The deacylation can also be carried out chemically as known in the priorart, for instance, via the formation of an iminochloride side chain, byadding phosphorus pentachloride at a temperature of lower than 10° C.and subsequently isobutanol at ambient temperatures or lower.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Fermentative production of phenylacetyl- 7-ADCA

P. chrysogenum strain Panlabs P14-B10, deposited at CBS under theaccession number 455.95, is used as the host strain for the expandaseexpression cassette constructs.

The expression cassette used containing the expandase gene under the P.chrysogenum IPNS gene transcriptional and translational regulationsignals is described in Crawford et al. (supra). Transformation andculturing conditions are as described in Crawford et al. (supra).Transformants are purified and analyzed for expression of the expandaseenzyme by testing their capacity to produce adipoyl-7-ADCA as describedby Crawford et al. (supra).

Adipoyl-7-ADCA producing transformants as for instance P. chrysogenumstrain PC100, deposited with the ATCC under number 74182 are inoculatedat 2.106 conidia/ml into a seed medium consisting of (g/l): glucose, 30;Pharmamedia (cotton seed meal), 10; Corn Steep Solids, 20; (NH₄)₂ SO₄,20; CaCO₃, 5; KH₂ PO₄, 0,5; lactose, 10; yeast extract, 10 at a pHbefore sterilisation of 5.6.

The seed culture (20 ml in 250 ml Erlemeyer closed with a cotton plug)is incubated at 25° C. at 220 rpm. After 48 hours, 1 ml was used toinoculate 15 ml of production medium consisting of (g/l): KH2PO₄, 0,5;K₂ SO₄, 5; (NH₄)₂ SO₄, 17,5; lactose, 140; Pharmamedia, 20; CaCO₃, 10;lard oil, 10 at a pH before sterilisation of 6.6.

After inoculation with the seed culture, 0.15-0.75 ml of 10%phenylacetic acid solution, adjusted to pH 7.0 with KOH, is added to thefermentation.

The production culture is inoculated at 25° C. at 220 rpm for 168 hoursin a 250 ml Erlemeyer flask closed with a milk filter. Evaporated wateris replenished every other day.

At the end of the production fermentation, the mycelium is removed bycentrifugation or filtration and penicillin G and phenylacetyl-7-ADCAare analyzed by HPLC.

EXAMPLE 2 Analysis of phenylacetyl-7-ADCA production

Fermentation products from transformed Penicillium strains were analyzedby high performance liquid chromatography (HPLC). The HPLC systemconsisted of the following Spectra Physics components: P1500 solventdelivery system, AS 1000 injector, UV1000 variable wavelength detector(set at 214 nm) and a ISM 100 integrator or similar. The stationaryphase was a Chrompack Chromspher C18 column. The mobile phase consistedof 75% phosphate buffer pH 2.6 and 25% acetonitril. The products werequantitated by comparison to a standard curve of phenylacetyl-7-ADCA andpenicillin G. The identity of the phenylacetyl-7-ADCA was established by600 MHz NMR of a deutero-chloroform solution obtained by acid extractionof the culture filtrate. The resonances of the phenylacetyl-7-ADCA inthe acid extract proved to be identical with those of a syntheticsample.

We claim:
 1. A method to prepare and obtain7-amino-desacetoxycephalosporanic acid (7-ADCA) which method comprisesa)fermenting a strain of Penicillium chrysogenum in a culture medium,wherein said strain has been modified to contain an expandase gene undertranscriptional and translational regulation of fungal expressionsignals; b) adding to said culture medium phenylacetic acid or a salt orester thereof so as to result in production of penicillin G which isexpanded to form phenylacetyl-7-ADCA; c) recovering thephenylacetyl-7-ADCA from said culture medium; d) deacylating therecovered phenylacetyl-7-ADCA to form 7-ADCA; and e) recovering 7-ADCA.2. The method of claim 1 wherein said 7-ADCA is recovered in crystallineform.
 3. The method of claim 1 wherein the recovering performed in stepe) is performed by filtration.
 4. The method of claim 1 wherein saidrecovering in step c) is by filtration of said culture medium followedby extracting the filtrate with an organic solvent immiscible with waterat a pH of less than about 4.5, followed by back-extracting with waterat a pH of 4-10.
 5. The method of claim 1 wherein the expandase gene isisolated from Streptomyces clavuligerus or Nocardia lactamdurans.
 6. Themethod of claim 2 wherein the expandase gene is isolated fromStreptomyces clavuligerus or Nocardia lactamdurans.
 7. The method ofclaim 3 wherein the expandase gene is isolated from Streptomycesclavuligerus or Nocardia lactamdurans.
 8. The method of claim 4 whereinthe expandase gene is isolated from Streptomyces clavuligerus orNocardia lactamdurans.